EMBO/SourceData huggingface dataset:
Figure 3F, The location diagram shows 4C enrichment around the negative control and ADGRG6 probe. Representative images of nuclei stained with DNA FISH probes for SNP rs1666389 (green) and negative control (red) or ADGRG6 (red) in H460 and A549 cells. The numerical points in the figure show the percentage of colocation in seven slides (with a total of 80 nuclei). Arrows point to the colocation signals. Nuclei with green or red positive signal are listed in the counting range. Values shown are means ± SD. Scale bars, 5 μm. ns, nonsignificant, *p < 0.05, **p < 0.01, unpaired two-tailed Student's t test.G, Expression of ADGRG6 at the protein levels in H460 and A549 cells.H, eQTL analysis demonstrating the correlation between the rs1663689 genotype and the expression of ADGRG6 in the clinical patient cohorts. The p value of the linear model was assessed by the Kruskal-Wallis test. Values shown are means ± SD.
Figure 4A, Protein expression of ADGRG6 or Actin after ADGRG6 knockdown in T/T cells and overexpression in C/C cells.Boyden chamber assay (C) of ADGRG6-knockdown T/T cells and ADGRG6-overexpressing C/C cells. Values shown are means ± SD obtained from one experiment. Scale bars, 200 μm. Three independent experiments were performed. **p < 0.01, ****p < 0.0001, unpaired two-tailed Student's t test.
Figure 5A, Immunoblot showing the expression of PKA substrate phosphorylation, PKA and Actin in the indicated cells and genotypes and the indicated cells treated with H89.BrdU incorporation assay (B) of the indicated cells after treatment with H89. Scale bars, 200 μm. Values shown are means ± SD obtained from one experiment. Three independent experiments were performed. ns, nonsignificant, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.D, Representative images and quantification of patient-derived organoids treated with 20 μM H89 and controls. The mean ± SD represents 5 visual fields from one experiment. Scale bars, 20 μm. ns, nonsignificant, ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.
Figure 1Graphs show changes in cytotoxicity between WT and GR T24 and UMUC3 cells exposed to increasing concentrations of GEM, and WST assays were performed 48 hr after treatment (n=3, biological replicates).Major metabolites altered in the glycolytic pathway in CR cells and GR cells compared to WT cells. The data are shown as the means ± SDs (n=4) and were analyzed by Student's t test.Western blot analysis of GLUT1, PKM2, LDHA, and β-actin compared among WT, CR, and GR cells.
Figure 2Levels of endpoint metabolites of the purine synthesis pathway in GR cells relative to those in WT cells as determined by CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. n.s.= non-significant   Levels of endpoint metabolites of the pyrimidine synthesis pathway in GR cells relative to those in WT cells as determined by CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01.  Western blot analysis of Hif-1α, G6PD, TIGAR, TKT, CTPS1, and β-actin in WT and GR cells under normoxia and hypoxia (left: T24 cells, right: UMUC3 cells).CE-MS-based metabolite detection for diphosphate nucleosides in WT and GR cells. The data are shown as the means ± SDs (n=3, biological replicates). The levels in GR cells are presented relative to those in WT control cells and analyzed by Student's t test. ***p<0.001. N.D.= non-detectable.
Figure 3Levels of intermediate metabolites of the tricarboxylic acid cycle (TCA) cycle in GR cells relative to those in WT cells based on targeted CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01.Relative 2-hydroxyglutarate (2-HG) production level based on targeted CE-MS metabolomics comparing among WT and GR cells (left panel: T24, right panel: UMUC3). The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01. Relative mRNA expression levels of IDH2 and EGLN1 genes compared between WT and GR cells (left panel: T24, right panel: UMUC3). The expression levels were analyzed by Student's t test and plotted relative to expression levels in WT cells. The data are shown as the means ± SEs (n=3, biological replicates. *p<0.05, ***p<0.001.Western blot analysis of IDH2, PHD2, and β-actin in WT and GR cells (left panel: T24, right panel: UMUC3).
Figure 4Western blot analysis of Hif-1α, GLUT1, G6PD, TIGAR, TKT, CTPS1, PKM2, LDHA, and β-actin after transfection with NTC and IDH2-targeting (siHIF1A#1 and #2) siRNAs (left: T24GR, right: UMUC3GR).Relative mRNA expression levels of IDH2, GLUT1, G6PD, TIGAR, TKT, CTPS1, PKM2, and LDHA in cells transfected with siIDH2#1 and #2 compared with cells transfected with siNTC (upper: T24GR, lower: UMUC3GR). The data are shown as the means ± SEs (n=3, biological replicates) and were analyzed by Student's t test, and plotted relative to expression levels in GR cells transfected with siNTC. *p<0.05, **p<0.01, ***p<0.001.   Western blot analysis of IDH2, PHD2, HIF-1α, G6PD, GLUT1, TIGAR, TKT, CTPS1, PKM2, LDHA, and β-actin after transfection with NTC and IDH2-targeting (siIDH2#1 and #2) siRNA (left: T24GR, right: UMUC3GR).  Graph showing the viability of GR cells exposed to various concentrations of GEM for 48 hr after transfection with NTC and IDH2-targeting (siIDH2#1 and #2) siRNA (left: T24GR, right: UMUC3GR) The data are shown as the means ± SDs (n=3, biological replicates).
Figure 5Relative mRNA expression levels of IDH2, EGLN1, Hif-1α, G6PD, TIGAR, TKT, and CTPS1 between J82 wild-type cells (J82WT) and J82 cells with IDH2 overexpression (J82-IDH2ox cells). The data are shown as the means ± SEs (n=3, biological replicates). The expression levels were analyzed by Student's t test and are plotted relative to the expression levels in WT cells. *p<0.05, **p<0.01, ***p<0.001.Western blot analysis of IDH2, PHD2, Hif-1α, G6PD, TIGAR, TKT, CTPS1, and β-actin in J82WT and J82-IDH2ox cells.Graph showing the viability of J82WT and J82-IDH2ox cells exposed to various concentrations of GEM for 48 hr. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. ***p<0.001.
Figure 6Changes in cytotoxicity between WT and GR T24 and UMUC3 cells exposed to increasing concentrations of CDDP for 48 hr. The data are shown as the means ± SDs (n=3, biological replicates).Immunohistochemistry (IHC) staining of Ki-67 in UMUC3WT and GR cells exposed to CDDP/GEM. Ki-67 staining quantitation of UMUC3WT and UMUC3GR cells exposed to CDDP/GEM are shown as bar graph The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by one-way ANOVA with the Bonferroni test. ** p<0.05, ***p<0.001. n.s.= non-significantNADPH/NADP ratios of GR cells transfected with NTC, IDH2 siRNA#1, and siRNA#2 (left: T24GR, right: UMUC3GR). The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. ** p<0.01.
Figure 7Graph showing the viability of GR cells exposed to various concentrations of GEM for 48 hr in combination with DMSO or 10 µM AGI6780 (upper: T24GR, lower: UMUC3GR). The data are shown as the means ± SDs (n=3, biological replicates).IHC staining for IDH2, Ki-67, CAIX, TIGAR, TKT, and CTPS1 in groups with GEM alone, AGI6780 alone, and the combination of GEM and AGI6780.
Figure 8Representative immunostaining of IDH2, CAIX (surrogate marker of Hif-1α), TIGAR, TKT, and CTPS1 in surgical specimens from upper tract urothelial carcinoma (UTUC) patients. The upper panel represents the immunostaining of metabolic enzymes of early-stage UTUC tumors, whereas the lower panel represents the images of invasive UTUC tumors. The power fieldKaplan-Meier curves showing the cancer-specific survival (CSS) of 74 UTUC patients treated with GC adjuvant chemotherapy. Metabolic enzymes were classified according to the results of receiver operating curve (ROC) analysis. Data were compared with the log-rank test.Kaplan-Meier method showing the CSS of 32 MIBC patients treated with GC chemotherapy. The cutoffs for metabolic enzyme levels were determined according to the results of ROC analysis. Data were compared with the log-rank test.
Figure 1Western blotting in the WT, Trim71 knockout (Trim71_KO), and the Trim71 mutant (R783H) mESCs. The signals from the pluripotency factors Nanog and Oct4 were obtained through short (~10 seconds) exposure.Colony formation assay for mESCs. The mESCs were cultured in 15%FBS + Lif for 7 days, and the resultant colonies were fixed and stained for AP.Exit pluripotency assay for mESCs. The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i+Lif medium for 5 days. The resultant colonies were fixed and stained for AP.Western blotting of pluripotency factors during EB formation. The signals from the pluripotency factors Nanog and Oct4 were obtained through long (5-10min) exposure.Immunofluorescence (IF) staining showing the expression level of Rex1 in mESCs cultured in the stemness (2i + Lif) and differentiating (15% FBS - Lif) conditions.Relative intensity of IF signals from individual cells in experiment associated with E. The results represent the means (± SD) of three independent IF stainings.Expression of neural lineage markers during mESCs neural differentiation.Expression of neural lineage markers during mESCs neural differentiation.IF staining of Nestin. The quantifications represent the means (± SD) of three independent experiments.
Figure 2Distribution of the WT Trim71 and the R783H Trim71 mutant binding regions in the mouse genome. In each CLIP-seq, there are two biological replicates. The binding regions present in both of the biological replicates are used for the analysis.CLIP-qRT-PCR for the identified target mRNAs of the Trim71 mutant R783H. The results represent the means (±SD) of three independent experiments.UCSC genome browser snapshot for the CLIP-seq data from Trim71 and the Trim71 mutant R783H in the Lsd1 locus. The red box indicates the binding region of the Trim71 mutant R783H. The inputs are from the size-matched input samples in the CLIP-seq analysis.
Figure 3B. A representative set of Western blots in the WT and the R783H Trim71 mESCs. C. Quantification of the target proteins in the WT and the R783H Trim71 mESCs. Data information: The results represent the means (± SD) of three independent experiments. One-Way ANOVA was used to determine the significance of the difference, *p<0.05. The Western blots listed in B is the Biological Replicate 1
Figure 4Western blotting in the WT and the Trim71 (R783H) mESCs.Quantification of Lsd1 protein and mRNA levels. Gapdh and 18S rRNA were used for normalization in protein and mRNA quantifications, respectively.Polysome analysis in the WT and the Trim71 (R783H) mESCs.Quantification of the indicated mRNA distribution in the RNP, 80S, and polysome fractions from the WT and the Trim71 (R783H) mESCs.Western blotting in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).Quantification of Lsd1 protein and mRNA in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).Polysome analysis in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H). Quantification of the indicated mRNA distribution in the RNP, 80S, and polysome fractions from the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).
Figure 5Deletion of the Trim71 mutant R783H binding site in Lsd1 mRNA's 3'UTR.CLIP-RIP followed by qRT-PCR to examine mRNAs associated with the Trim71 and the Trim71 mutant R783H in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs. The mRNA signals from the E14 mESCs were set as 1 for relative comparison.Western blotting in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs.Quantification of Lsd1 protein and mRNA in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs. Beta-Tubulin and 18S rRNA were used for normalization in the protein and mRNA quantification, respectively.Exit pluripotency assay for mESCs.Representative Western blotting and quantification of pluripotency factors during EB formation.Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation.
Figure 6Western blotting in the WT and the Trim71(R783H) mESCs with dox-inducible expression of Lsd1-GFP.Exit pluripotency assay for mESCs.Representative Western blotting and quantification of pluripotency factors during the monolayer differentiation of mESCs.Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation.
Figure 7Representative Western blotting and quantification in the WT and the R783H/+ mESCs.Exit pluripotency assay for the WT and the R783H/+ mESCs.Representative Western blotting and quantification of pluripotency factors in differentiating mESCs.Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation.Western blotting in the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.Exit pluripotency assay for the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.Representative Western blotting and quantification of pluripotency factors during the differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.Representative Western blotting and quantification of neural lineage markers during the neural differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.
Figure 2A, RT-qPCR (n = 3-12 biological replicates) analyses of CGN expression in different mouse organs at P21.B. Western blot analyses of CGN expression in different mouse organs at P21.G. Whole mount immunofluorescence of CGN expression in P3, P14 and P21 mouse cochlea coimmunostained with cuticular plate marker LMO7 (G). CGN expression levels at the cuticular plates were normalized to LMO7 immunofluorescent signals (n = 130-193 hair cells from 3-4 cochleae). Data information: Data are presented as mean ± SEM; one-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.
Figure 3B. MDCK cells expressing WT or mutant CGN were immunolabeled with CGN or Flag antibodies.D. RT-qPCR of CGN expression in transfected MDCK cells (n = 3 biological replicates). Data information: Data are presented as mean ± SEM; unpaired Student's t-test was used in (D). ns, not significant, P > 0.05.
Figure 4A-C. DPOAE thresholds (A), ABR thresholds (B) and ABR P1 amplitudes (C) of the 2-month old control (Cgnfl/fl) and Cgn-cKO (Cgnfl/fl;Pou4f3creER/+) mice. Control mice (circle), n = 16 mice (female = 8, male = 8); Cgn-cKO mice (square), n = 19 mice (female = 11, male = 8). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.D-I. Growth curves of ABR P1 amplitudes at different cochlear frequencies (5.6 - 32 kHz) of the 2-month old control and Cgn-cKO mice. Control mice (circle), n = 16 mice (female = 8, male = 8); Cgn-cKO mice (square), n = 19 mice (female = 11, male = 8). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.J. Whole mount immunofluorescence of the organ of Corti from 2-month old control and Cgn-cKO mice. Phalloidin (F-actin, magenta); Myosin7a (hair cells, green). White arrows label the lost OHCs.
Figure 5C. Whole mount immunofluorescence of CGN expression in organ of Corti from P21 CgndelG mouse.D-I. DPOAE thresholds (D, E), ABR thresholds (F, G), and ABR P1 amplitudes at 32 kHz (H, I) of 2-month old (D, F, H) or 6-month old (E, G, I) CgndelG mice. Control Cgn+/+ mice (circle), n = 3 mice (2-month, female = 1, male = 2) or 15 mice (6-month, female = 4, male = 11); CgndelG/+ mice (triangle), n = 8 mice (2-month, female = 4, male = 4) or 20 mice (6-month, female = 9, male = 11); CgndelG/delG mice (square), n = 7 mice (2-month, female = 4, male = 3) or 16 mice (6-month, female = 6, male = 10). Cgn+/+ vs CgndelG/delG, * (red); Cgn+/+ vs CgndelG/+, * (green). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.J. Whole mount immunofluorescence of the organ of Corti from 7-month old CgndelG mice. White arrows indicate lost OHCs by F-actin labeling (Phalloidin).K. Percentage of OHC loss in 7-month old CgndelG mice (n = 3-8 biological replicates from 3 cochleae). Data information: Data are presented as mean ± SEM; two-way ANOVA was used * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.
Figure 6F. Percentage of OHC loss in noise exposed CgndelG mice (n = 5-9 cochleae). Data information: Data are presented as mean ± SEM; one-way ANOVA was used in (F). ** P < 0.01, *** P < 0.001 and **** P < 0.0001.
Figure 7Whole mount immunofluorescence with LMO7 antibody on 2-month old CgndelG mice at 32 kHz cochlear regionWhole mount immunofluorescence with Spectrin β II antibody on 2-month old CgndelG mice at 32 kHz cochlear region
Figure 8A. Low-magnification SEM images of hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. B. High-magnification SEM images of IHC hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. C. High-magnification SEM images of OHC hair bundles from 2-month old Cgn+/+ and CgndelG/delG mice. Shown are hair bundles of the second-row OHCs (OHC2).
Figure 1B Image sequence from confocal time lapse shows the early steps in neuronal differentiation. Two transient basal protrusions are extended along the basal surface of the neural tube (6h 26 to 14h 01) and then retracted (15h 31 to 17h 31). The apical attachment is also retracted (17h 31 to 19h 53; e.g. -1h 30,) before the axon is extended (20h 38 to 21h 50). Main images are maximum projections and insets are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.C Image sequence from confocal time lapse shows a neuron before, during and after axon initiation. Prior to axon initiation, the neuron extends multiple small, transient pre-axonal protrusions (-2h to -20m). The nascent axon is extended (0h) and maintained for a short period (0h to 30m) before axon growth begins (45m to 2h 20). Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.D Plots showing axon position on the soma (e.g. asterisk in Figure 1C 0h) relative to the cell centroid at 0,0 for dorsal and transverse view (n = 86 cells from 8 experiments). Axon position is not random (dorsal view P < 0.001, mean = 95.3o; transverse view P < 0.001, mean = 148.9o). Data analysed using Moore's modification of the Rayleigh's test.F Lateral and transverse reconstructions of DoLA neuron at the time of axon initiation (0h) and during axon growth (4h). Data information: All scale bars = 10 µm.G-M Plots showing axon position on the soma relative to cell centroid at 0,0 in dorsal and transverse view for DoLA (G; n = 7 cells), CiA (H; n = 10 cells), CiD (I; n = 3 cells), VeLD (J; n = 5 cells), CoSA (K; n = 11 cells), CoB (L; n = 6 cells) and UCoD (M; 15 cells) neuronal subtypes.
Figure 2A Image sequence showing centrosome position of a neuron labelled with membrane and centrosome labels during apical process retraction. The centrosome is retracted close to the tip of the apical process. Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.B (i) Image sequence showing a neuron labelled with a membrane marker during apical retraction without any observable abscission event (n = 64/72 cells from 6 experiments). (ii) High resolution images of boxed section in (i). Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.C (i) Low magnification overview of the spinal cord of a cilium reporter line, Tg(actb2:arl13b-GFP) from a single confocal slice. (ii) High resolution image sequence of boxed section in (i). One GFP-labelled cilium moves from apical surface towards the basal surface of the spinal cord (n = 13 cilia from two experiments). Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.D (i) Low magnification overview of the spinal cord of a Tg(actb2:arl13b-GFP) embryo labelled with centrin-RFP from a single confocal slice. (ii) Image sequence of boxed section in (i). A cilium and centrosome move together from apical surface towards the basal surface of the spinal cord (n = 2 cells from one experiment). Insets show high magnification of cilium-centrosome pair. Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.E Graph showing length of cilium as it moves from apical surface (0%) to close to basal surface (100%. Cilium length did not change (P > 0.05 for n = 6/7 cilia from two experiments; non-linear regression).F Distance between centrosome and cilium in Tg(actb2:arl13b-GFP) embryos fixed and processed for immunohistochemistry against GFP to label the cilium and γ-tubulin to label the centrosome. No difference was found in the distance between the two organelles when close to the apical surface or away from the midline (n = 50 cells per position from two experiments; P = 0.8279; midline mean = 0.4956, s.d = 0.1125; away from midline mean = 0.5004, s.d = 0.1077; Student's unpaired t-test). Bars show mean and standard deviation.
Figure 4A Image sequence from confocal time lapse of a neuron labelled with a membrane marker and lifeact-Ruby before, during (0m) and after axon initiation. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.B Graph showing time (minutes) between actin accumulation and nascent axon initiation (n = 15 cells from 7 experiments). Bars show mean and standard deviation.C Image sequence from confocal time lapse of a neuron labelled with lifeact-Ruby and EB3-GFP before, during (0m) and after axon initiation. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.D Image sequence from confocal time lapse of a neuron labelled with a membrane marker and Kif5c560-YFP before, during (0m) and after axon initiation. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.E Three time points from confocal time lapse of a triple labelled neuron before, during (0m) and after nascent axon initiation. Images to left show distribution sequence of lifeact-Ruby, kif5c560-YFP and EB3-GFP individually. Dual channel merges to the right show relative locations of pairs of fusion proteins. Images to right are maximum projections of transverse reslices of confocal z-stacks. Data information: All scale bars = 10 µm.